This presentation entitled "Golden rice" explains the needs for golden rice development, Biotechnological manipulations in metabolic pathways for GR-1 and GR-2 development and finally it also detailed with the associated ethical issues.

1. PROFESSOR JAYASHANKAR TELANGANA STATE
AGRICULTURAL UNIVERSITY
College of Agriculture, Rajendranagar, Hyderabad- 500030
Presented by,
Ajay Kumar Chandra
RAM/14-97
M.Sc. (Ag) Mol. Biology & Biotechnology

2. What …… we going to discuss today?
• Introduction and Why....Rice?
• Golden Rice… Project?
• -Carotene Pathway: Problem in Plants.
• Production of Golden rice: Genetic modification
• The Golden Rice: Solution
• How to make golden rice?
• Golden Rice-1: limits
• Golden Rice 2
• Controversy: Golden Rice…..Is it worth the risks?
• Golden Rice: A boon or bane?
• Clinical trials / food safety and nutrition research
• Conclusion
• Reference

3. Why....Rice?
 Milled rice is provitamin-a-free.
 Symptoms of a provitamin-a-free
diet
• Night-blindness
• Xerophthalmia
• Fatal susceptibility to childhood diseases
(e.g. measles) and general infections
(diarrhoea, respiratory diseases).
 Epidemiology
• 124 million children are deficient in
vitamin-A.
• 1-2 million deaths annually (1-4 years).
• 0.25-0.5 million deaths (5-10 years)
UNICEF.
• For many countries, the
infrastructure doesn’t exist to deliver
vitamin pills.
• Improved vitamin-A content in major
staple consumed crops an attractive
alternative to fight micronutrient
deficiencies.
 Solution
• In 2005, 190 million children and 19 million pregnant women, in 122 countries,
were estimated to be affected by VAD.

4. Golden Rice… Project?
• Idea first discussed at international conference at IRRI
in Philippines in 1984.
• 1999 Team of scientists, including Ingo Potrykus, Swiss
Federal Institute of Technology, successfully genetically
engineer rice to produce carotenoids, precursors to
Vitamin-A.
• The hype begins: 2000 Time magazine cover story:
“This rice could save a million kids a year.”
• May 2000: Adrian Dubock (Zeneca, now Syngenta):
“One month delay = 50,000 blind children [a] month.”
• June 29, 2000 US special Congressional Forum, “Can
Biotechnology Solve World Hunger?” Invitation stated,
“ ‘golden rice’, which has been modified to include
certain vitamins……….Is already saving the sight of
thousands of children in the poorest parts of Asia.”

5. -Carotene Pathway: Problem in
Plants IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
 -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Problem:
Rice lacks
these enzymes
Normal
Vitamin A
“Deficient”
Rice

6. Production of Golden rice: Genetic
modification
• Golden Rice technology is based on the simple principle that rice plants
possess the whole machinery to synthesis of β-carotene, and while this
machinery is fully active in leaves, parts of it are turned off in the grain
endosperm.
• By adding only two genes, a plant phytoene synthase (psy) and a bacterial
phytoene desaturase (crt I), the pathway is turned back on and β-carotene
consequently accumulates in the grain.
• Golden rice was created by transforming rice with only two β-carotene
biosynthesis genes:
1). psy (Phytoene synthase) from daffodil (Narcissus pseudonarcissus)
2). crtI (Carotene desaturase) from the soil bacterium Erwinia uredovora

7. Figure: Gene construct used to generate Golden Rice-1.
RB, T-DNA right border sequence; Glu, rice endosperm-specific glutelin promoter;
tpSSU, pea ribulose bis-phosphate carboxylase small subunit transit peptide for chloroplast
localisation; crtI Carotene desaturase from the soil bacterium Erwinia uredovora; nos,
nopaline synthase terminator; Psy, phytoene synthase gene from Narcissus pseudonarcissus
(GR1) or Zea mays (GR2); Ubi1, maize polyubiquitin promoter; Pmi, phosphomannose
isomerase gene from E. coli for positive selection (GR2); LB, T-DNA left border sequence.
• Lycopene is then cyclized to beta-carotene by the endogenous cyclase in
Golden Rice.
(The insertion of a lcy (lycopene cyclase) gene was thought to be
needed, but further research showed it is already being produced in wild-type
rice endosperm.)

8. • The psy and crtI genes were transformed into the rice nuclear genome and
placed under the control of an endosperm-specific promoter, so they are
only expressed in the endosperm.
• The exogenous lcy gene has a transit peptide sequence attached so it is
targeted to the plastid, where geranylgeranyl diphosphate formation occurs.
• The end product of the engineered pathway is lycopene, but if the plant
accumulated lycopene, the rice would be red.
Recent analysis has shown the plant's endogenous enzymes process the
lycopene to β-carotene in the endosperm, giving the rice the distinctive yellow
color for which it is named..
• The original golden rice was called SGR1, and under greenhouse conditions
it produced 1.6 µg/g of carotenoids.
Genetic modification ......

9. The Golden Rice: Solution
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
 -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Daffodil gene
Single bacterial gene;
performs both functions
Daffodil gene
-Carotene Pathway Genes Added
Vitamin A
Pathway
is complete
and functional
Golden Rice
Presence of pro-vitamin -A gives rice grains a yellowish-orange color, thus, the name
‘Golden Rice.’

11. Golden Rice-1: limits
• Originial Golden Rice (GR1) does not produce enough ß-carotene (Provitamin A); it produces
“only 1.6 μg/gm of carotenoids; a child would have to eat more than 10kg/day to
get sufficient dose”.
• Unexpected effect: GR1 was supposed to produce lycopene (as in tomatoes) and so be
bright red; instead, it produced ß-carotene due to unexpected metabolic pathway.

12. Golden Rice 2
• In 2005, Syngenta, produced a variety of golden rice called "Golden Rice 2".
• They combined the phytoene synthase(psy) gene from maize with carotene
desaturase (crt1) from the original Golden rice-1.
• Both genes are under endosperm specific promoter control and the
mannose act as selectable marker.
• Golden rice 2 produces 23 times more carotenoids than golden rice (up
to 37 µg/g), and preferentially accumulates β-carotene (up to 31 µg/g of the
37 µg/g of carotenoids).
• To receive the Recommended Dietary Allowance (RDA), it is estimated that
people who eat about 75g of golden rice per day.
• In June 2005, researcher Peter Beyer received funding from the Bill and
Melinda Gates Foundation to further improve golden rice by increasing
the levels of or the bioavailability of pro-vitamin A, vitamin E, iron, and
zinc, and to improve protein quality.

13. Figure: Gene construct used to generate Golden Rice-2.
RB, T-DNA right border sequence; Glu, rice endosperm-specific glutelin promoter;
tpSSU, pea ribulose bis-phosphate carboxylase small subunit transit peptide for chloroplast
localisation; crtI Carotene desaturase from the soil bacterium Erwinia uredovora; nos,
nopaline synthase terminator; Psy, phytoene synthase gene from Narcissus pseudonarcissus
(GR1) or Zea mays (GR2); Ubi1, maize polyubiquitin promoter; Pmi, phosphomannose
isomerase gene from E. coli for positive selection (GR2); LB, T-DNA left border sequence.
Transform long grain rice variety (Kaybonnet) Sugar selectable marker
619 individual GM rice plants
Screen for seed colour, gene copy number, fertility
Select 6 “Golden Rice 2” events for further screening and development
Golden Rice-2

14. Controversy: Golden Rice…..Is it
worth the risks?
 Health
• May cause allergies or fail to perform desired effect.
• Supply does not provide a substantial quantity as the recommended daily
intake.
 Environment
• Loss of Biodiversity. May become a super weed and endanger the existence
of natural rice plants.
• Genetic contamination of natural, global staple foods.
• Gene flow from GM to non- GM field crops.
 Culture
• Some people prefer to cultivate and eat only white rice based on traditional
values and spiritual beliefs on Veg. or Non-Veg.
• Intervention in “Gods creation”.
• Critics of genetically engineered crops have raised
various concerns.

15. Golden Rice: A boon or bane?
• An early issue was that “golden rice originally did not have sufficient vitamin A”.
• “The speed at which vitamin A degrades once the rice is harvested”, and
“how much remains after cooking are contested.”
• Greenpeace opposes the use of any patented genetically modified organisms in
agriculture and opposes the cultivation of golden rice, claiming “it will open the
door to more widespread use of GMOs.”.

16. • Other groups argued that a varied diet containing foods rich in beta carotene such as
sweet potato, leafy green vegetables and fruit would provide children with sufficient
vitamin A.
• Keith West of Johns Hopkins, Bloomberg School of Public Health countered that
“foodstuffs containing vitamin A are either unavailable, or only available at certain
seasons, or that they are too expensive for poor families in underdeveloped
countries”.
Vandana Shiva, an Indian anti-GMO activist,
argued “the problem was not the plant per se,
but potential problems with poverty and loss of
biodiversity”.
Shiva claimed “these problems could be
amplified by the corporate control of agriculture.”
Golden Rice: A boon or bane?

17. Clinical trials / food safety and
nutrition research
• IRRI also assures the public that “Golden Rice” will be available to farmers and
consumers only after it has been determined to be safe for humans, animals, and the
environment and authorized for propagation and consumption by the appropriate
regulatory authorities. Therefore, “Golden Rice”, if and when released, will be deemed
to be as safe as other rice.
• According to the information reported by the WHO, genetically modified products that
are currently on the international market have all passed risk assessments conducted by
national authorities." These assessments found no risk to human health”.
• The Food Allergy Resource and Research Program of the University of Nebraska (2006)
that showed “the proteins from the new genes in Golden Rice did not show any
allergenic properties”.
• In 2009, research results of a clinical trial of Golden Rice with adult volunteers from the
USA were published in the American Journal of Clinical Nutrition. It concluded that "β-
carotene derived from Golden Rice is effectively converted to vitamin A in humans is a
safe".
• In August 2012, Tufts University and others published new research on Golden Rice in
the American Journal of Clinical Nutrition showing that “the β-carotene produced by
Golden Rice is as good as β- carotene in oil at providing vitamin A to children”.

18. “Public sentiment is everything. With public sentiment, nothing can
fail. Without it, nothing can succeed.” – Abraham Lincoln.
Conclusion

19. Reference
• BOOKS REFFERED:
GENE CLONING AND DNA ANALYSIS: AN INTRODUCTION - T. A. Brown;
5th edition; Blackwell publication.
GENE CLONING: PRINCIPLES AND APPLICATIONS - Julia lodge, Pete Lund
& Steve Minchin; Taylor & Francis Publishing Group.
GENE VIII- Benjamin Lewin; Pearson Education, Inc.
• LITERATURE CITED:
Al-Babili S, Hoa TTC, Schaub P (2006) Exploring the potential of the bacterial carotene
desaturase CrtI to increase the β-carotene content in Golden Rice. J Exp Bot 57:1007-1014.
Paine JA, Shipton CA, Chaggar S, Howells RM, Kennedy MJ, Vernon G, Wright SY,
Hinchliffe E, Adams JL, Silverstone AL, Drake R (2005) A new version of Golden Rice
with increased provitamin A content. Nature Biotechnology 23:482-487
Al-Babili S, Beyer P (2005) Golden Rice – five years on the road – five years to go? TRENDS in
Plant Science 10:565-573.